ADAM17 (a disintegrin and metalloprotease 17) is a cell surface metalloprotease with vital roles in regulating the EGF-receptor as well as TNFa signaling. EGFR-ligands and TNFa are made as membrane- anchored precursors that must be proteolytically released or ?shed? to activate the soluble signaling molecule. Mice lacking ADAM17 resemble mice lacking the EGFR, providing genetic evidence for the essential role of ADAM17 in EGFR signaling. Moreover, inactivation of ADAM17 in myeloid cells protects from septic shock in mice, which is caused by the release of soluble TNFa from myeloid cells. These functions establish ADAM17 as an important potential target for the treatment of EGFR- and TNFa-dependent pathologies such as cancer and autoimmune diseases. ADAM17 activity is highly regulated and is influenced by numerous signaling pathways. How these pathways functionally intersect with ADAM17 and how ADAM17 is activated are key questions that are the main focus of my lab. Previously, we had found that ADAM17 is regulated by its transmembrane domain (TMD), which ultimately led us to identify the seven-membrane spanning iRhoms1 and 2 (iR1, iR2) as novel regulators of ADAM17. We showed that iR2 controls the function of ADAM17 in immune cells and that inactivation of iR1 and iR2 abolishes all functions of ADAM17 in mice, thereby providing biochemical, cell biological and genetic evidence that iR1 and iR2 are the long sought-after regulators of ADAM17. The main goals of the current proposal are to understand how iR1 and 2 integrate, interpret and execute the signals that drive the activation of the ADAM17/EGFR- and ADAM17/TNFa signaling pathway. In the next 5 years, we will explore the biosynthesis and regulation of the iRhom/ADAM17 complexes and how they target substrates in a selective manner. We know that the cytoplasmic domain of ADAM17 is required to stabilize the protein, most likely so that it can assembly with an iRhom in the endoplasmic reticulum (ER). We would like to identify the factors that stabilize ADAM17 and the sites in ADAM17 and the iRhoms that promote their interaction. Once the iRhoms have assembled with ADAM17, they move to the cell surface. We will use mutant iRhoms that are retained in the ER to identify binding partners that regulate their export from the ER. We will also explore how the substrate selectivity of iRhoms is determined at the molecular level. Finally, ADAM17 is unique in that it responds rapidly to several different signaling pathways, a property that is thought to be essential for its role in skin and intestinal barrier protection. We will study the molecular details of the on/off switch for ADAM17, and generate mice that express a constitutively active ADAM17 that cannot be activated to determine how important this unique property of ADAM17 is in vivo. Together, our planned studies will resolve the most pressing current questions regarding the regulation of ADAM17, a major cellular sheddase that is critical for TNFa and EGFR signaling and a target for treatment of cancer and autoimmune diseases, by its recently discovered upstream regulators iR1 and iR2.